INVESTIGADORES
MORENO Silvia Margarita
artículos
Título:
Characterization of yeast pyruvate kinase 1 as a protein kinase A substrate and specificity for the phosphorylation site sequence in the whole protein.
Autor/es:
PORTELA, P., MORENO, S. AND ROSSI, S.
Revista:
BIOCHEMICAL JOURNAL
Referencias:
Año: 2006 vol. 396 p. 117 - 126
ISSN:
0264-6021
Resumen:
Pyk1 (pyruvate kinase 1) from Saccharomyces cerevisiae was
characterized as a substrate for PKA (protein kinase A) from
bovine heart and yeast. By designing Pyk1 synthetic peptides containing
potential PKA sequence targets (Ser22, Thr94 and Thr478)
we determined that the peptide S22 was a substrate for PKASaccharomyces cerevisiae was
characterized as a substrate for PKA (protein kinase A) from
bovine heart and yeast. By designing Pyk1 synthetic peptides containing
potential PKA sequence targets (Ser22, Thr94 and Thr478)
we determined that the peptide S22 was a substrate for PKA22, Thr94 and Thr478)
we determined that the peptide S22 was a substrate for PKA
in vitro, with a Ksp∗ (specificity constant) 10-fold and 3-fold
higher thanKemptide for bovine heart and yeast PKArespectively., with a Ksp∗ (specificity constant) 10-fold and 3-fold
higher thanKemptide for bovine heart and yeast PKArespectively.
In vitro phosphorylation of the Pyk1 S22A mutant protein was
decreased by as much as 90% when compared with wild-type
Pyk1 and the Pyk1 T94A mutant. The Ksp∗ values for Pyk1
and Pyk1 T94A were the same, indicating that both proteins are
phosphorylated at the same site by PKA. Two-dimensional PAGE
of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation
prevented the formation of one of the Pyk1 isoforms.We conclude
that in yeast the major PKA phosphorylation site of Pyk1 is
Ser22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. Thephosphorylation of the Pyk1 S22A mutant protein was
decreased by as much as 90% when compared with wild-type
Pyk1 and the Pyk1 T94A mutant. The Ksp∗ values for Pyk1
and Pyk1 T94A were the same, indicating that both proteins are
phosphorylated at the same site by PKA. Two-dimensional PAGE
of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation
prevented the formation of one of the Pyk1 isoforms.We conclude
that in yeast the major PKA phosphorylation site of Pyk1 is
Ser22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. The% when compared with wild-type
Pyk1 and the Pyk1 T94A mutant. The Ksp∗ values for Pyk1
and Pyk1 T94A were the same, indicating that both proteins are
phosphorylated at the same site by PKA. Two-dimensional PAGE
of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation
prevented the formation of one of the Pyk1 isoforms.We conclude
that in yeast the major PKA phosphorylation site of Pyk1 is
Ser22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. TheKsp∗ values for Pyk1
and Pyk1 T94A were the same, indicating that both proteins are
phosphorylated at the same site by PKA. Two-dimensional PAGE
of Pyk1 and Pyk1 S22A indicates that in vivo the S22A mutation
prevented the formation of one of the Pyk1 isoforms.We conclude
that in yeast the major PKA phosphorylation site of Pyk1 is
Ser22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. Thein vivo the S22A mutation
prevented the formation of one of the Pyk1 isoforms.We conclude
that in yeast the major PKA phosphorylation site of Pyk1 is
Ser22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. The22.
Phosphorylation of Ser22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. The22 leads to a Pyk1 enzyme that is more
active in the absence of FBP (fructose 1,6-bisphosphate). The
specificity of yeast and mammalian PKA towards the S22 peptide
and towards whole Pyk1 proteinwas measured and compared. The
Ksp∗ for the S22 peptide is higher than that for Pyk1, indicating
that the peptide modelled on Pyk1 is a much better substrate than
Pyk1, regardless of which tissue was used as the source of PKA.
However, the Km of Pyk1 protein is lower than that of the better
substrate, the S22 peptide, indicating that ground-state substrate
binding is not the major determinant of substrate specificity for
PKA.sp∗ for the S22 peptide is higher than that for Pyk1, indicating
that the peptide modelled on Pyk1 is a much better substrate than
Pyk1, regardless of which tissue was used as the source of PKA.
However, the Km of Pyk1 protein is lower than that of the better
substrate, the S22 peptide, indicating that ground-state substrate
binding is not the major determinant of substrate specificity for
PKA.Km of Pyk1 protein is lower than that of the better
substrate, the S22 peptide, indicating that ground-state substrate
binding is not the major determinant of substrate specificity for
PKA.